DE19629543C2 - Immunoassay for the detection of anti-B. burgdorferi antibodies and methods for serodiagnosis in Lyme disease, diagnostic agents and test kits for performing the methods - Google Patents

Abstract

The following are claimed: (1) a diagnostic reagent comprising ≥ 4 antigens that are derived from a Borrelia afzelii membrane fraction and are selected from P870, P412, P402, P365, P315, P310, P280, P250, P230 and P180; (2) a diagnostic reagent comprising ≥ 4 OspC antigens derived from Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34; (3) monoclonal antibodies to immunoreactive P870, P412, P402, P365, P315, P310, P280, P250, P230 and P180 antigens derived from a membrane fraction of B. afzelii, and (4) immunoreactive P870, P412, P402, P365, P315, P310, P280, P250, P230 and P180 antigens derived from B. afzelii strain Orth.

Description

The invention relates to an immunoassay for the detection of anti-B. burgdorferi antibodies in a sample. Particularly The invention relates to diagnostic agents containing as Reagent components discriminating antigens derived from B. afzelii and / or OspC antigen from selected RFLP types by Borrelia burgdorferi s. l. as well as a procedure for Serodiagnosis of Lyme disease with an improved Specificity and sensitivity and for differential detection of antibodies against various serotypes of B. Burgdorferi.

Lyme disease (LB) is one of the most common of ticks transmitted infectious diseases of humans. she is a multisystemic disease caused by the spirochete Borrelia burgdorferi is evoked. B. Burgdorferi belongs to the family of spirochetes, i. e. helical Bacteria of 8-30 µm in length, which come from an outer shell, the endoflag cells in the periplasm and the protoplast cylinder consist. To the human pathogenic representatives of the spirochetes In addition to B. burgdorferi, relapsing fever borrelia belong (e.g. recurrentis), the causative agent of syphilis (Treponema e.g. B. T. palladium) and the leptospires. To B. burgdorferi sensu lato belong to the representatives of the species B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. japonica and B. andersonii, with all three former representatives in Europe are widespread, while B. burgdorferi sensu stricto is predominant in North America.  

The close immunological relationship of the pathogens makes it difficult the serological detection of antibodies in syphilis and Lyme disease with the tests available so far.

The clinical spectrum of Lyme disease includes three syndromes (EM, ACA and MPN-GBB), which have long been described as independent clinical pictures. Similar to syphilis, Lyme disease is divided into three different stages, which occur in a different period after infection with the pathogen:
Stage I (days to weeks): erythema migrans (EM), lymphadenosis cutis benigna (LCB)
Stage II (weeks to months): secondary EM, LCB, meningitis, encephalitis, myelitis, arthralgia
Stage III (months to years): ACA, encephalomyelitis, arthritis.

Lyme disease can be prevented by administering antibiotics such as Penicillin G, tetracyclines, erythromycin or Cephalosporins. Heals in some cases Lyme disease often spontaneously in the early stages out, however, late manifestations are also not locked out. There is also a clinical cure Antibiotic therapy for late manifestation only in a few To achieve cases. Lyme disease should therefore, if possible be diagnosed early.

With the variety of symptoms of Lyme disease is a Evidence of infection through clinical diagnosis in connection with serological methods inevitable.

With Lyme disease, however, not all three are always Go through stages; smooth transitions and symptom-free Intervals are also possible. There are stadiums too The disease can be skipped in any of the  manifest three phases for the first time. This fact leads Again and again to misjudgments, as often accepted becomes that the typical manifestation of Lyme disease, namely the EM must be run through before a later one Manifestation can occur. Because the diverse Appearance of Lyme disease often others Similar forms of disease, it can occur in purely clinical Diagnoses come to misjudgments. This danger increases even more so if the patient's medical history does not Evidence of a previous tick bite.

The antibody detection (IgM, IgG) is in the serum and at neurological manifestations also from the cerebrospinal fluid carried out.

Serological examinations showed Patients with EM predominantly positive IgM findings, in neurological manifestations predominantly positive IgG Findings. In the late manifestations of ACA and Lyme arthritis the IgG titer is regularly increased and IgM only in Exceptional cases detectable (Wilske et al. 1984. Infection 12: 331-337, Herzer et al. 1986. Zbl. Bakt. Hyg. A 263: 268- 274). Exposed persons, such as forest workers, have a hand increasing age often a significantly increased Antibody level.

The serological findings depend on the stage of Illness, the duration of the symptoms and possibly one antibiotic therapy. So is the antibody detection with previously available test systems for EM only in 20-50%, at neurological manifestations in 50-90% and in ACA and Arthritis possible in 90-100% of cases. Increased antibody Titres are not always clear diagnostically as it is both a clinically manifest infection and can act as an epidemic.

So there is only one thing left for the definite confirmation of the diagnosis the path, the pathogen from the various organs or  Isolate body fluids or specific ones Evidence of immune reactions against B. burgdorferi. A Pathogen detection from patient material is particularly in the Early stage (EM), where antibody detection is common negative is recommended. The cultivation of borrelia however requires a complex medium, a lot of experience in Cultivation and is very time consuming since up to five weeks are required for the pathogen detection.

With the use of the sensitive polymerase chain reaction (PCR) could diagnose the direct pathogen become usable. Unfortunately, the PCR has so far only been possible in Animal experiments are used successfully; for one however, it is not yet used for diagnostic purposes in humans evaluated enough. For example, Schwartz et al. (1992. J. Clin. Microbiol. 30: 3082-3088) after that the PCR compared to the conventional cultivation technology only slightly better Results. The sensitivity, measured on sera from Early patients, using PCR, were 59-62% during the Cultural evidence was successful in 57% of the cases. The high sensitivity However, the PCR brings with it that - as with all other methods - wrong results are also possible here (Berger, 1994. Dermatol. Clin. 12: 19-24).

After the direct detection methods are too time consuming, serological tests are usually carried out, whereby opt for routine immunofluorescence assay (IFA), the ELISA and immunoblot analysis to have. Since the IFA is very subjective and labor intensive, he was replaced by the ELISA. For serological diagnosis Lyme borreliosis is therefore the ELISA and Western blot most widely used test systems. The ELISA test has the Advantage that it can be automated and therefore good for "screening" large sample quantities is suitable. On the issue of Specificity proved to be better with the Western blot, although this test is more labor intensive and at the  Evaluation requires a lot of experience. Especially in the very The sensitive early phase of Lyme disease is the Western blot awarded a higher detection rate (Aguero-Rosenfeld et al., 1993. J. Clin. Microbiol. 31: 3090-3095; Stiernstedt et al., 1991. Scand. J. Infect. Dis. Suppl. 77: 136-142; Sentence, 1992. Switzerland. med. Weekly 122: 1779-1791; Schmitz et al., 1993. Eur. J. Clin. Microbiol. Infect. Dis. 12: 419-424). An important difference between the two test systems is that that more native epitopes can be determined with the ELISA than the Western blot.

The disadvantage of a whole cell lysate ELISA test is that that it only indicates whether antibodies with those in the ELISA used antigens react or not. It will be there on the one hand, conformative epitopes are detected, but it is on the other hand it is not possible to distinguish whether it is this reaction by a specific reaction or by a Cross reaction. This often leads to wrong positive results. A problem of the specificity of the tests are broad cross reactions of the pathogen B. burgdorferi too other bacterial pathogens, such as oral spirochetes, others Borrelia and especially T. palladium, the causative agent of Syphilis. Since the antigens are generally derived from lysates of the entire pathogen, antibodies are also against "common antigens" recorded. In order to achieve the To reduce cross-reactivity will be investigated Sera therefore often with T. phagedenis, B. hermsii or E. coli absorbed. These methods are said to be the most important cross-reacting antibodies are removed from the sera.

The disadvantages of the Western blot are that the most conformative epitopes cannot be detected and so far no standardization is possible.

Given the advantages and disadvantages of both Test systems have been proposed more and more, the ELISA as  "Screening assay" and the Western blot as "confirmatory Assay "(Stanek, 1991. Infection. 19: 263-267; Rose et al., 1991. Pediatrics. 88: 465-470; Grodzicki et al., 1988. J. Infect. Dis. 157: 790-796). Although these suggestions accordingly already commercially available combination test kits at the Basis of the ELISA and the Western blot are available no test of sufficient specificity and sensitivity (Bakken et al., 1992. JAMA. 268: 891-895). Christenson et al. (1991. J. Clin. Lab. Anal. 5: 340-343) found that the Sensitivity of the ELISA test systems in the early phase of the Lyme Lyme disease in 57-71% and in late-stage patients in 86-100% lies.

The results of a serological test are strong depending on which test antigen is used. Most of them Tests are called whole B. burgdorferi cell lysates or antigens Whole cell ultrasonificates used. Since most published tests only organisms of the species B. burgdorferi sensu stricto, especially strain B31, use the Heterogeneity of the individual species, however, again and again is emphasized (by Dam et al., 1993. Clin. Infect. Dis. 17: 708- 717; Wilske et al., 1994. Med. Microbiol. Immunol. 183: 43- 59), results need to be specific regarding the specificity Antigens should always be interpreted carefully. Another one Problem when using whole cell lysates, in particular with western blot, the overload of antigens is in certain areas, thereby not being properly decided which antigen has reacted.

Because of the problems associated with whole cell lysates, was attempted to use only those Borrelia antigens in serological Consider tests that are as specific as possible and occur regularly. So far however, no antigen can be found that meets these requirements is sufficient (sentence, 1992. Switzerland. med. Wochenschr. 122: 1779-1791).  

There were some common surface proteins be found with an immunogenic character (Osp A, OspC); it However, it was shown that this (i) from the individual tribes are expressed to different degrees and (ii) also in different serotypes within the individual subspecies be divided. According to Wilske et al. the lipoprotein OspA in separate at least 7 different serotypes (Wilske et al., 1993. J. Clin. Microbiol. 31: 340-350). In WO 93/04175 there are further descendants of B. burgdorferi OspA serotypes described. These antigens show with those hitherto known little homology. On their Usability in the treatment or diagnosis of Lyme Lyme disease compared to the known is in WO 93/04175 also received. Owing to The lipoprotein OspC can be analyzed at least in RFLP analyzes Separate 35 serotypes (Livey et al., 1995. Mol. Microbiol. 18: 257-269). In the various investigations showed that a reaction with OspA is specific, nevertheless it occurs rarely and if so, only in the Late phase of Lyme disease (Engstrom et al., 1995. J. Clin. Microbiol. 33: 419-427).

Especially in the early phase of the disease, antibodies become against OspC. However, studies have shown that OspC Has sequence homologies with antigens from B. hermsii and cross-reactions between the antigens are observed (Carter et al., 1994. Infect. Immun. 62: 2792-2799). OspC there is still a big one in serological diagnostics Importance because it is a very good immunogen and one Antibody response can be detected. Wilske et al. (1994. Med. Microbiol. Immunol. 183: 43-59) found that when testing recombinant OspC the IgG Antibodies, but not the IgM antibody, strongly species shows specific behavior. IgM antibodies therefore appear Conservative epitopes from recombinant OspC detect too can. Gerber et al. (1995. J. Infect. Dis. 171: 724-727) therefore proposed recombinant OspC for the diagnosis of use early Lyme disease. They found in various test systems (whole cell lysate ELISA, immunoblot and IgM rOspC ELISA) with sera from patients with a clear EM a sensitivity up to 46% and an almost 100% specificity. However, you note that only the Evidence must be provided that IgM antibodies from  Patients with no or other clinical manifestations early Lyme disease with rOspC respond to the To confirm sensitivity of this test.

The fact that there are several subtypes at OspC and OspA there, complicates the use of these proteins in one diagnostic kit because theoretically everyone different subtypes would have to be united. Because sera of Lyme disease patients mostly antibodies against only have one of the three known species, Theisen et al. (1993. J. Clin. Microbiol. 31: 2570-2576), in one serodiagnostic test OspC antigen from all three B. burgdorferi groups (B. burgdorferi sensu stricto, B. garinii, B. afzelii).

In addition to OspA and OspC, the proteins P100 (identical to P83 or E90), 41 kD and P39 as immunodominant proteins described and used in a diagostic. Reactions against P100 can be found in early-stage patients, however Corresponding antibodies are usually only in the further Course of disease formed. Antibodies against the 41 kD Antigens appear very early, but it turned out that Reactions against this antigen itself are not specific for B. burgdorferi, because cross-reactions with others Organisms such as E. coli or Treponemen have been observed (Luft et al., 1993. Res. Microbiol. 144: 251-257; Ditton et al., 1992. FEMS Microbiol. Lett. 94: 217-220). With the P39 is a protein that is mainly in the Early phase of the disease of antibodies is detected (Ma et al., 1992. J. Clin. Microbiol. 30: 370-376; Aguero- Rosenfeld et al., 1993. J. Clin. Microbiol. 31: 3090-3095). So far, all five proteins mentioned have also been identified recombinant way and against different Patient and control sera tested (Wilske et al., 1994. Med. Microbiol. Immunol. 183: 43-59; Fawcett et al., 1993. J. Rheumatol. 20: 734-738).  

The results of previous studies of various However, test systems led to the conclusion that an diagnostic kit based on serology not only can be based on a test antigen and that sensitivity through the use of antigens from different species can be increased by B. burgdorferi sensu lato (Wilske et al. 1994. Proceedings of the VI International Conference on Lyme Borreliosis, Bologna June 19-22, 179-182). Wilske et al. therefore combined recombinantly produced immunodominants Antigens (P100, p41, OspA, OspC, p41i) from representative Strains of B. burgdorferi sensu lato in an immunoblot. she found that the specificity through the use of this recombinant proteins is increased significantly, however Sensitivity clearly in comparison to whole cell lysates is lower and the detection rate of specific IgM or IgG antibodies with the respective specific protein varied. When using recombinant antigens there were also significant differences between the single species. This is how P100 reacted in a study Sera from neuroborreliosis patients vary, each after which species it was expressed (Wilske et al., 1994. Med. Microbiol. Immunol. 183: 43-59).

Due to the strong variation in the reaction pattern of strains of different origins (skin etc.) or countries (North America or Europe) the question arose to what extent the Heterogeneity of European strains the serological Tests influenced. In particular it should be mentioned that in commercial test kits predominantly B. burgdorferi antigens sensu stricto strain B31 can be used.

A number of antigens in the Western blot Molecular weight range of 19 kD, 22 kD, 29 kd, 34 kD, 35 kD, 60 kd, 66 kD and 74 kD were also identified (Coleman et al., 1992. J. Infect. Dis. 165: 658-666; Wallich  et al., 1993. Infect. Immune. 61: 4158-4166; Lam et al., 1994. Infect. Immune. 62: 290-298; Luft et al., 1991. J. Immunol. 146: 2776-2782; Norris et al., 1992. Infect. Immune. 60: 4662- 4672; Bergström et al., 1992. Scand. J. Infect. Dis. 24: 181- 188; Scopio et al., 1994. J. Bacteriol. 176: 6449-6456). EP 0 465 204 discloses B. burgdorferi B31 antigenic proteins with a molecular weight of 25, 28, 38, 44, 48, 52, 54, 58, 60, 68, 80 and 90 kD (+ -3kd) used in a diagnostic Kit can be used. WO 90/04411 describes immunologically active fractions of B. burgdorferi B31 and ACA, the antigens with different molecular weights include.

As mentioned above, this occurs both when using recombinant antigens as well as whole cell lysate antigens Difficulties with the sensitivity or specificity of the Antigen reactions. Especially when using Whole cell lysates were used for the evaluation of immunoblots tried through suitable evaluation criteria that Specificity and sensitivity of the test procedure too increase. For example, Dressler et al. (1993. J. Infect. Dis. 167: 392-400) for the evaluation of Western blots in the Sero diagnosis so-called discriminatory test criteria developed, with a positive result by the occurrence of at least 2 out of 8 possible bands in the Molecular weight range of 18, 21, 28, 37, 41, 45, 58 and 93 kD in IgM and at least 5 out of 10 possible bands in Range from 18, 21, 28, 30, 39, 41, 45, 58, 66 and 93 kD at IgG is displayed in the immunoblot.

Ma et al. (1992. J. Clin. Microbiol. 30: 370-376) found in Immunoblot significant antigens from B. burgdorferi sensu stricto (strain B31) in the molecular weight range from 94, 83, 75, 66, 60, 55, 46, 41, 39, 43, 31, 29, 22 and 17 kD.  

Engstrom et al. (1995. J. Clin. Microbiol. 33: 419-427) used B. burgdorferi for serological tests sensu stricto strain 297 and struck as positive criteria for IgM an immune reaction with 2 of 3 proteins (24, 39 and 41 kD) and for a positive IgG with 2 out of 5 proteins (20, 24, 35, 39 and 88 kD). However, it is unanimous of the Experts pointed out that for the assessment of the Serodiagnosis in Lyme disease, especially in Evaluation of immunoblots, no satisfactory criteria for To be available.

For the problem that patients are positive clinical picture but negative serology, have so far been unable to find suitable procedural criteria To be defined. There are also none at the moment satisfactory diagnostic interpretation for those people which is not a clinical picture, but a positive serology have (inapparent or silent infection). Although at several serological methods are currently known there is still no test that is satisfactory Possesses sensitivity and specificity and is also able to on a serological basis between early and late manifestation to distinguish. Furthermore, there is still no serological one Test system that makes a distinction between the different species and serotypes of B. burgdorferi allows an infection. So far there is none standardized Western blot available improved detection of Lyme disease allowed. this applies especially with regard to the existing problems with methodological variants, the variability of B. burgdorferi Strains and the complexity of the antigens.

There is therefore an urgent need for an improved one serological test with a high sensitivity and Specificity for the determination of a seroconversion in Lyme Lyme disease, as well as a determination of the species of the infection,  to initiate therapeutic measures in good time. It there is still an urgent need for deployment of B. burgdorferi specific antigens for development a diagnostic test for Lyme disease that if possible Contains antigens that (1) of all pathogenic B. burgdorferi Strains are expressed, (2) having high antigenicity detectable antibodies in the early stage of the immune response and (3) no cross-reactivity with non-B. burgdorferi have specific antigens. In the Development of a diagnostic test is therefore the selection of the Antigens that have a high sensitivity and specificity for the Have evidence of early infection from vitally important.

The aim of the present invention is therefore a improved method according to the type mentioned above To provide which is an increased sensitivity and Has specificity, is easy to standardize and Detection of Lyme Disease and how to create one Serodiagnosis also in an early stage of infection as well as Determination of the serotype of the causative infection used can be. Such a test should also be an option provide forecasts for symptoms of the disease to set up.

The above object is achieved according to the invention by Providing a diagnostic agent which as reacting components at least 4, preferably at least 6, most preferably at least 8, of a membrane fraction of B. afzelii-derived antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 includes. It is preferred that the invention diagnostic agents all of the above Includes antigens. If the invention diagnostic agents not all of the antigens mentioned involves or if some of the antigens by others  exchanged, it may still be sufficient specific test for Lyme disease. A however, the best result is when all are present of the antigens mentioned.

Surprisingly, it was found that the one Membrane fraction from B. afzelii strain Orth contained Antigens with a molecular weight of 87.0 kD, 41.2 kD, 40.2 kD, 36.5 kD, 31.5 kD, 31.0 kD, 28 kD, 25.1 kD, 23 kD and 18 kD represent discriminating antigens that are specific with anti-B. burgdorferi antibodies react and none Cross reactivity with others, not Lyme disease have specific antibodies. By the increased Specificity and sensitivity of the in the antigens contained in diagnostic agents is therefore a Improved detection of seroconversion if suspected Lyme disease possible.

The discriminating antigens were identified by an analysis of the Molecular weights of the proteins of a membrane fraction of B. afzelil. A membrane fraction II from B. afzelii prepared, which is characterized in particular by the fact that they have a balanced ratio of different antigens contains, causing an overload of proteins with the same or similar molecular weight, as in whole cell lysates occurs, is avoided. A preparation from B. Burgdorferi cells consist of several hundred different ones Proteins. In one preparation Membrane fractions from B. burgdorferi were included in this Invention identified a total of 63 different proteins. The proteins of the membrane fraction were previously accurate determined, standardized conditions in one Gel electrophoresis system separated and the molecular weights certainly. Only the improved analysis conditions allowed  a clear and reproducible assignment of proteins to an appropriate molecular weight.

In a further step, the proteins were analyzed using a Series of monoclonal antibodies identified so that a distinction between already known and not yet identified antigens was possible. It was found that three of the B. afzelii proteins are equivalent to already known antigens of other B. burgdorferi strains are: P870 is equivalent to protein P100 or E90 from PKo strain; P365 is equivalent to protein 35E and P251 is equivalent to OspC.

To identify such antigens that are present in a A distinction between cross-reactive and immune response allow specific antigens, was a panel of sera of Lyme disease patients with different types of syndrome, of syphilis patients and a blood donation control group against proteins separated by molecular weight Membrane fraction II from B. afzelii tested. The evaluation the immunoblots allowed the identification of so-called discriminating antigens specific to anti-B. burgdorferi antibodies react and their reaction with an antibody that may be present in a sample the sample was seroconverted for Lyme disease identify. It has been shown to be discriminatory identified antigens of membrane fraction II from B. afzelii in an immune reaction characterized by a special high specificity and sensitivity to B. burgdorferi Label antibodies.

According to an embodiment of the present invention the diagnostic agent comprises a membrane fraction of B. afzelii, preferably selected from the group of B. afzelii strain Orth and strain H15. The B. afzelii strain Orth is part of the German Microorganism Collection (DMS)  under number 11048 and strain H15 under number 11047 deposited in accordance with the Budapest Treaty.

According to a further embodiment of the present Invention contains the diagnostic agent as Reagent components the recombinant antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180, the Antigens are derived from membrane fraction II antigens from B. afzelii with a molecular weight of 87.0 kD, 41.2 kD, 40.2 kD, 36.5 kD, 31.5 kD, 31.0 kD, 28 kD, 25.1 kD, 23 kD and 18 kD. The advantage of using recombinant Antigens reside in that (i) only specific binding of serum antibodies with these antigens is possible; (ii) it is possible to mix mixtures of homologous antigens to produce different serotypes or species; (iii) a simplification and / or standardization of the test is made possible; (iv) higher sensitivity is achieved; and (v) the specific antigen in sufficient amount in the test is available.

The recombinant antigens can be obtained using known genetic engineering processes are produced. The for the Production of the recombinant antigens using methods include steps such as isolating the antigens of the corresponding molecular weight from the SDS gel, manufacture of monoclonal antibodies, such as by Lane et al. (1985. J. Immunol. Method, 81: 223), expression Screening of a gene bank from B. afzelii with the monoclonal Antibodies and identify and isolate the positive Clones that express the recombinant antigen. From cells transfected with the antigen-expressing clone can then remove the antigen using standard methods be isolated and cleaned. The purified antigens can be used as reagent components in the invention diagnostic means to provide evidence of Lyme disease can be used.  

Genetic engineering can be used with all common prokaryotic and eukaryotic expression systems, such as e.g. B. with plasmids that express in host cells such as E. coli, B. subtilis or yeast, especially in P. pastoris allow to be carried out.

According to a further embodiment, this contains Diagnostic agents according to the invention as reagent components those derived from a membrane fraction II from B. afzelii purified antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180. The cells of a selected one Strain of B. afzelii, in particular of B. afzelii strain Orth, are lysed according to known methods that Membrane fraction II by selective centrifugation from the other cell components separated and the proteins by means of a detergent, preferably a non-denaturing one Detergent, extracted from the membrane. The solubilized Proteins are then chromatographed Methods such as ion exchange chromatography or HPLC, cleaned.

The diagnostic agent according to the invention contains the Reagent components P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 preferably isolated or spatially separated from each other and immobilized on a solid support in front. If the diagnostic agent according to one Embodiment is a membrane fraction of B. afzelii, the proteins are extracted using gel electrophoretic methods, such as SDS-PAGE, separated by molecular weight and then on a solid support, e.g. B. on a membrane or a filter, preferably a nitrocellulose membrane, immobilized.

According to a special embodiment, the proteins a membrane fraction or a mixture which has been purified  Contains antigens in a 1-lane SDS gel with a Track width of z. B. 8 cm, separated and on a membrane transferred. The transfer of proteins can be done through known methods, such as diffusion technology or Electrical transfer.

Those so bound to a solid support Reagent components of the diagnostic agent then for performing a Western blot, preferably a modified Western blot in the form of a Surfblot®, used. Testing antibodies against separated and trans-blotted antigens of a chosen B. Burgdorferi strain is made according to the present invention referred to as vertical analysis. The membrane with the bound antigens are either in strips cut, the stripes marked and the individual stripes incubated with a sample, or the membrane is placed in a Surfblot® apparatus clamped, which means about 30 different samples simultaneously with the diagnostic Funds can be brought into contact.

Are purified antigens or in the diagnostic agent genetically engineered recombinant antigens as Reagent components are used, so they are isolated present antigens directly to a solid support, selected from plates, strips, membranes, filters, paper, Film or pearls, immobilized. For this, the isolated and optionally purified antigens in a suitable Buffer suspended and a desired aliquot of the suspension tied to the carrier.

According to a further embodiment, the according to the Purification isolates existing reagent components in one desired ratio mixed and the mixture, in essentially consisting of the purified B. afzelii Membrane proteins P870, P412, P402, P365, P315, P310, P280,  P251, P230 and P180, via gel electrophoresis after the Molecular weight separated and the antigens attached to a solid Carrier, preferably a membrane or a filter bound. The carrier-bound reagent components are used for one Vertical analysis for the detection of B. burgdorferi antibodies used.

According to a further embodiment, the and are isolated purified antigens for the production of a line Blot used. For this, an aliquot of a suspension containing a discriminating antigen, as a line, if necessary with the help of a surfblot apparatus, on the Carrier applied and immobilized.

According to a further embodiment, they are isolated antigens present on the surface of microtiter plates bound, the individual reagent components preferably be applied in separate depressions of the plate. It can but also a mixture of all antigens on the surface be bound in a plate trough. Testing the samples can then be carried out according to known ELISA test procedures , aliquots of a sample to be tested, for example a human serum for each reagent component given and then a chromogenic substrate reaction is carried out.

According to the present invention, this is carried out the immunoassay of the vertical analysis preferably as a Western Blot or ELISA. However, others in the state of the art Technically known immunassays, such as an enzyme, lumino, Fluoro or radioimmunoassay to perform the be used according to the inventive method.

Are used to produce the diagnostic Used with purified antigens Concentration of the in the Western blot, in the ELISA or a  other immunoassay components present be standardized because the antigen concentration beforehand determined, if necessary in any ratio mixed, and can be applied to the solid support.

If appropriate, contains the diagnostic according to the invention Agent as further reagent components at least one OspC Antigen, preferably at least 4, particularly preferred at least 10, most preferably at least 15, im essentially derived from Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34.

For an optimal test, it is preferred that the diagnostic agents according to the invention as further Reagent components all of the OspC antigens of the above mentioned Borrelia RFLP types are added. It is also possible that in the diagnostic agent according to the Invention of one or more of these antigens is absent or are replaced by others.

The diagnostic agent of the present invention In particular, the invention is used to carry out a Immunoassays for the detection of anti-B. burgdorferi antibodies used.

Another aspect of the present invention includes one Test kit for the detection of B. burgdorferi antibodies in one Vertical analysis containing a diagnostic tool of Type, reagents and solutions for Detection of an antigen / Antibody complex, an evaluation system to create a serodiagnosis and instructions as well as if necessary a Surfblot® device.  

Is the diagnostic agent according to a special one Embodiment as a surf blot membrane before, so can test up to 30 different samples at the same time. There own equipment for carrying out a surfblot is necessary, the test kit according to the invention contains if necessary a Surfblot® or an analog working equipment. Contain reagents and solutions in the test kit according to the invention, buffer such. B. TBS-Tween® 20 or PBS-Tween-20®, enzyme-conjugated antibodies, Enzyme substrate, positive and negative references, Reagent controls and control strips. Preferably the test kit contains a foil template, the corresponding one Markings on the running position of the discriminatory Has antigens, and an evaluation system.

According to a further embodiment, the Test kit according to the invention optionally a diagnostic Agent containing at least one OspC as reagent components Antigen, preferably at least 4, particularly preferred at least 10, most preferably at least 15, the essentially of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 is (are) derived. These reagent components can also be be bound to a solid support.

So far, the experts have proposed OspC antigens, derived from one representative of the species of B. burgdorferi sensu stricto, B. afzelii and B. garnii or ideally from all representatives of all known Serovar or use RFLP types in a diagnostic test. in the Within the scope of the present invention, OspC antigen, derived from all 35 known RFLP types against one representative panel of human sera tested, and it was surprisingly found that patient sera mostly with at least one of 20 selected RFLP Guys react. By using OspC antigen,  derived from 20 selected Borrelia RFLP types as Reagent components, in a diagnostic agent it is therefore possible for the first time with a serological method early immune response when infected with B. burgdorferi regardless of the serotype of the pathogen. Although known to be in an early stage of infection Antibodies against OspC are formed, it was previously due to the heterogeneity of OspC not always possible anti-OspC Detect antibodies in the immunoassay. Only through that Selection according to the invention of some OspC-RFLP types and their Use as reagent components in a diagnostic It is possible to create a system with high sensitivity and means To provide specificity, but at a minimum must be used on OspC antigen variants. This is especially with regard to economical production of a highly sensitive diagnostic test is crucial because theoretically all RFLP types should be included.

Another aspect of the present invention therefore relates also a diagnostic agent containing as Reagent components at least 4 OspC antigens, preferably at least 10, particularly preferably at least 15, essentially of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, that for an immunoassay for the detection of anti-B. Burgdorferi Antibodies and / or to determine the serotype of the Borrelia pathogen can be used. For one optimal test, it is preferred that the inventive diagnostic agents all of the OspC antigens of the above Borrelia RFLP types mentioned.

The diagnostic agent according to the invention is according to one Embodiment of the invention, a membrane fraction, which in essentially of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 is derived. Selected representatives of these RFLP types are  listed in Table 1. Of course, others can too Representatives of the RFLP types selected according to the invention, the by Livey et al. (1995. Mol. Microbiol. 18: 257-269) be described as the source of the reagent components be used.

Used for the preparation of the diagnostic agent Membrane fractions of the selected Borrelia RFLP types is used by individual representative tribes Membrane fraction isolated, the membrane proteins separated by gel electrophoresis, the proteins on a solid Carrier immobilized and the immobilized Reagent components for the detection of anti-B. burgdorferi antibodies or for the determination of the Serotypes used. OspC antigen can also pass through Isolation and purification of the proteins from Borrelia, as in EP 0 522 560.

According to a preferred embodiment, OspC antigen, derived from selected RFLP types from Borrelia genetically engineered and as a recombinant antigen in diagnostic means used. Livey et al. (1995. Mol. Micobiol. 18: 257-269) discloses the amino acid and Nucleotide sequences of OspC-RFLP types for the Production of the different OspC antigen variants using known genetic engineering methods can be used. Purified or recombinantly produced Osp-C antigen dissolved in a suitable buffer and on a support immobilized. If a membrane is used as a solid support, preferably a nitrocellulose membrane used, so it is applied to the membrane in the form of individual Drop of solution (dot blot) or as lines (line blot). The diagnostic agent thus produced can then corresponding to performing a horizontal western blot Analysis. Another version includes the application of the OspC antigens to a solid  Carrier substance, preferably in the form of microtiter plates, the individual reagent components each in one Plate trough can be immobilized. If necessary, too a mixture of some or all of the purified antigens the surface of a well are bound.

The present invention further encompasses the use a diagnostic agent containing as Reagent components OspC antigens, essentially derived Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, for the detection of B. burgdorferi antibodies and / or to determine the serotype of the Borrelia pathogen.

The diagnostic agent according to the invention is according to a Another aspect of the invention available in a test kit made of additional reagents and solutions for Performing a detection reaction of the formation of a Antigen / antibody complex and an evaluation system for Creating a serodiagnosis and instructions on how to do so.

As reagent components in the diagnostic agent are according to of the present invention from a membrane fraction of B. afzelii-derived antigens, essentially P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180, and / or OspC antigens, essentially derived from the Borrelia Types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, used, the Agents according to the invention each independently in separate immune test systems for the detection of B. Burgdorferi antibodies can be used.

According to the invention, the diagnostic test comprises at least 4 of the aforementioned antigens, preferably all or essentially all of the antigens mentioned.  

Both are preferred for establishing a serodiagnosis Test systems used because a positive proof under Circumstances is only possible with a test system.

According to one embodiment of the invention diagnostic agents containing discriminatory antigens, derived from membrane fraction II from B. afzelii in one Vertical analysis, preferably a Western surf blot analysis, and the diagnostic agent containing OspC-RFLP- Antigen variants in a horizontal analysis, preferably a line blot analysis. Through the combination of both analysis systems, vertical and horizontal analysis achieved a higher sensitivity and specificity, so that too such samples are classified as positive or negative can, for which no clear result has been possible so far was. The present method therefore has the advantage that in particular false negatives, but also by Cross-reaction false positive results can be excluded. However, it can be used for both Execution of the vertical analysis as well Horizontal analysis other known in the art Immunoassays, such as an enzyme, lumino, fluoro- or Radioimmunoassay.

According to a further embodiment, the cleaned Reagent components, both discriminatory Antigens as well as the selected OspC-RFLP types, in one ELISA test used by the isolated and purified Antigens separated, if necessary in one desired mixture, to the surface of individual Wells of microtiter plates bound and aliquots a sample to be tested with each reagent component in Be brought in contact. This makes it easy Combination of both immune test systems in the form of one ELISA tests possible.  

The present invention therefore also includes one Combination test kit from a diagnostic agent which essentially that of a membrane fraction II of B. afzelii derived antigens P870, P412, P402, P365, P315, Includes P310, P280, P251, P230 and P180 to perform a vertical analysis and a diagnostic tool, which, as reagent components, has at least one OspC antigen, preferably at least 4, particularly preferably at least 10, most preferably at least 15 OspC antigens derived Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 contains, for Performing a horizontal analysis to detect B. burgdorferi antibodies.

Another aspect of the present invention relates to one Immunoassay for the detection of anti-B. burgdorferi antibodies, being a diagnostic agent containing as Reagent components essentially from a membrane fraction B. afzelii-derived antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 and with a sample that is suspected, B. burgdorferi- Containing antibodies is incubated under conditions which allow the formation of an antibody / antigen complex and determining the presence of anti-B. Burgdorferi Antibodies by detection of a reaction product, consisting of an antigen / antibody complex, in particular the complex formation of an antibody with one of the Reagent components.

Another aspect of the present invention relates to one Immunoassay for the detection of anti-B. burgdorferi antibodies and / or determination of the serotype of the Borrelia pathogen, being a diagnostic agent containing as Reagent components at least 4 OspC antigens derived from Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, provided and  incubated with a sample that may have anti-B. burgdorferi contains antibodies and determine presence by anti-B. burgdorferi antibodies in the sample Detection of the formation of an antigen / antibody complex, in particular the complex formation of an antibody with a OspC-RFLP-type antigen component.

Samples that are tested with the method are human or of animal origin, especially blood, plasma, serum, CSF or synovial fluid.

So far has been used for serological diagnosis of Lyme disease proposed a two-test system (MMWR 1995, Vol. 44, 590-591), with preliminary evidence of infection with an EIA or IFA test should be done and the test is positive identified individuals confirmed by Western blot become. For those with the EIA or IFA test as negative identified individuals, it was not found necessary perform another test.

In the context of the present invention, 670 human sera tested and found that an anti-B. Burgdorferi specific immune response in both test systems (Vertical analysis or horizontal analysis), in some cases however, it can only occur in one of the immunoassays. It it was found that negative results of one sample in one Test, so-called non-responder, not necessarily are seronegative, since they are very good when tested in the second test can probably show an immune response. With that, the negative results of one test with either the second Test confirmed or demonstrated that the first test system gave a false negative result and the individuals belong to the responder group according to the second immunoassay. The test according to the invention is therefore both in the  Vertical analysis as well as horizontal analysis as "Confirmation Assay (Confirmation Test)" performed because false negative results excluded and positive ones Results are confirmed in another test. As above already mentioned, the previous diagnostic procedures sera classified as negative not in a confirmation- Assay further tested. It has, however, surprisingly found that a variety of samples that over previously known methods were assessed as negative by the method according to the invention identified as positive could become. This is particularly true in the early stages of the Infection is important because it is therapeutic Measures can be taken to make a manifestation the disease is prevented. Likewise, through the Methods according to the invention also test such samples as positive identified so far despite the late manifestation of the Disease have been defined as seronegative. The diagnosis of Patients with so-called "seronegative Lyme disease" has been very difficult so far and has been with conventional serological methods of determination are not possible. The method according to the invention therefore enables a sensitive determination of seroconversion in patients at an early stage of infection as well as in cases where So far no clear assignment in seropositive or seronegative was possible. It is also with the Methods according to the invention possible, Borrelia-specific Antibodies in samples of various origins, especially from different geographical regions, with very high sensitivity and specificity, since the Antigen components used in the immunoassay with almost react to all known serotypes of Borrelia.

Within the scope of the present invention, new ones were also created Seroconversion parameters defined, in particular in Borderline cases in which no clear assignment in positive or negative was possible, a better rating  enable. Measuring the parameters of seroconversion takes place according to the present invention in one standardized immune test system.

Seroconversion parameters to determine a positive Seroconversion was carried out according to the present invention as defined as follows:

I. Vertical analysis with subsequent horizontal analysis:

• a) Assigns a sample, for example a serum, an IgM or IgG reaction with at least one antigen reagent component in the vertical analysis, with the reagent component being one of a membrane fraction derived from B. afzelii P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 antigen , it is considered to be Lyme disease seroconverted.
• b) If a sample has no reaction according to a), it becomes in a horizontal analysis against a diagnostic agent, containing as reagent component OspC antigen, in essentially derived from Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, tested.

II. Horizon analysis with subsequent vertical analysis:

• a) If a sample exhibits an IgM or IgG reaction with at least an antigen reagent component in the horizontal analysis, wherein the reagent component is an OspC antigen, essentially derived from Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, so it is considered seroconverted with regard to Lyme disease.
• b) If a sample has no reaction according to a), it becomes in a vertical analysis against a diagnostic agent, containing as a reagent component one of a Membrane fraction derived from B. afzelii P870, P412, P402,  P365, P315, P310, P280, P251, P230 and P180 antigen, tested.

Is used in the sample tested according to I. b) or II. B) Antibody / antigen complex with an antigen component proven, the sample is considered to be in relation to Lyme Lyme disease as a seroconvert.

If the sample has no reaction according to I. b) or II. B), then if necessary, it will reappear after a certain period of time tested for an IgM or IgG reaction according to a) or b).

Only if in the sample according to a) or b), and if necessary after repeating the test, no IgM or IgG reaction is to be proven, the sample is clearly not considered seroconverted for Lyme disease.

The present invention therefore also includes a method for the serodiagnosis of Lyme disease, which comprises the following steps:

• a) Providing a diagnostic agent for Performing a vertical or horizontal analysis,
• b) Incubate a biological sample that may anti-B. burgdorferi contains antibodies with which diagnostic agents, and
• c) determining the presence of anti-B. Burgdorferi Antibodies in the sample by detecting the formation of a Antigen / antibody complex, the formation of a Complex between an antibody and a Antigen reagent component of the diagnostic agent for a Seroconversion is indicative of Lyme disease.

Tested samples containing antibodies in the Vertical analysis with one of the antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 from B. afzelii or in horizontal analysis with a selected OspC-  RFLP-type antigens form a complex, according to this Procedure classified as responder, i.e. as seroconverted. The samples, which are analyzed using an analytical method, e.g. B. one Vertical analysis are tested, if necessary Confirmation of the result of a second immunoassay, e.g. B. the Subjected to horizontal analysis, passing through the second test at the same time a statement about the serotype of the pathogen is made possible.

The sera with no antibody-antigen complex formation with the discriminating antigens of vertical analysis or can be determined in the horizontal analysis, apply in with regard to the immunoassay carried out as a non- Responders and will be used to verify the Result tested with another immunoassay. There possibly none in the early stages of the infection Antibodies against a discriminating antigen Membrane fraction II have been formed by B. afzelii, it is particularly important against non-responders in vertical analysis to test an antigen, in particular the antibodies that are in the early phase of the immune response is recognized. The Sample of a non-responder, tested with discriminatory Antigens, is therefore only after testing with selected OspC Antigens in horizontal analysis than in relation to Lyme Lyme disease is rated as seronegative or seropositive.

If for patient groups based on the medical history there is reasonable suspicion of a Borrelia infection, however with the method described above in none of the Immunoassays to detect seroconversion should be used Confirmation of the diagnosis after a specified period of time an immunoassay be carried out again because as is known, only about 4 weeks after the Borrelia Infection-specific antibodies must be detected.  

According to the invention, the method is preferably used for Serodiagnosis of Lyme disease and / or to predict the Disease syndrome after Borrelia infection by determination of the syndrome-associated antigen.

The immunoassay method according to the invention has in particular the advantages that it is (i) easy to manufacture and can be standardized, (ii) by the combination of two Immune test systems gives clear results, with both false negatives and false positives excluded (iii) a large number of samples simultaneously and with reproducible result can be tested and (iv) for the user is easy to handle and evaluate.

Within the scope of the present invention, the as discriminatingly identified antigens monoclonal Antibodies produced. The present invention encompasses hence monoclonal antibodies against the immunological active antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 of a membrane fraction from B. afzelii Strain Orth. These monoclonal antibodies can be used in one Immunassay are used in which the antibodies as Reagent components for the detection of Borrelia antigens in a sample can be used.

Another use of the monoclonal antibodies is in using them to purify the immunologically active antigens of borrelia. To do this, the antibodies with known methods on a solid support, for example a column matrix coupled, and the bound antibodies used for affinity chromatography. By means of this Affinity chromatography can identify specific antigens Whole cell lysates from Borrelia or from recombinant Cell cultures expressing the antigen are purified.  

In the context of the present invention, new antigens of Membrane fraction II from B. afzelii strain Orth identified and characterized by their molecular weight. The invention therefore also includes immunologically active proteins from B. afzelii Stamm Orth P870, P412, P402, P365, P315, P310, P280, P230 and P180 with a molecular weight of 87.0 kD, 41.2 kD, 40.2 kD, 36.5 kD, 31.5 kD, 31.0 kD, 28.0 kD, 23.0 kD and 18.0 kD.

Brief description of the figures:

Fig. 1 Gel electrophoretically separated by molecular weight membrane fraction II of B. afzelli strain Orth. All antigens that were included in the analysis are labeled with their corresponding molecular weight (in kDa). Those antigens that were also identifiable with known monoclonal antibodies are given short descriptions in the figure (Osp = "Outer Surface Protein", E = "Equivalent to" with the following number indicates the approximate molecular weight of the corresponding antigen in the homologous strain kD on).

Fig. 2 Number of determinable by SDS gel electrophoresis potential antigens of the membrane fraction II of Borrelia strains.

Fig. 3 Surfblot a membrane fraction II of B. afzelli strain Orth, wherein the colored colloidal gold stripes those antigens are labeled (with molecular weights or abbreviations, see above), which proved to be at the vertical analysis (VA) as significant.

Fig. 4 Result of a vertical analysis of a selection of 37 human sera, tested against membrane fraction II from B. afzelii strain Orth.

Fig. 5 Summary of identified as differentiating and non-redundant antigens of the membrane fraction II of B. afzelii strain Orth.

Fig. 6 Horizontal analysis of the anti-OspC immune response of Lyme disease patients.

Figure 7 Specificity of anti-OspC immune response from human sera.

Fig. 8 Summary of the reaction pattern of the IgG and IgM response of 37 human sera from Lyme borreliosis patients after horizontal analysis against OspC antigen variants.

Fig. 9 Horizontal analysis of human sera against recombinant OspC antigens in a line blot

Fig. 10 Summary of the results of the horizontal and vertical analysis of 190 patient sera from Åaland, Finland.

Fig. 11 Summary of the results of the horizontal and vertical analysis of 37 representative patient sera, Åaland, Finland

Fig. 12 Comparison of the vertical and horizontal analysis results from representative patient sera.

Fig. 13 Comparison of the results of the seropositivity criteria of human sera from 3 different study areas

Fig. 14 detection rates of sera that can be detected either only with vertical analysis or only with horizontal analysis.

Fig. 15 detection rate of the horizontal analysis in non responder sera that still exhibited at Ausstestung in the vertical analysis neither an IgG an IgM response.

Fig. 16 Comparison of the results of Phase I patients in the study of sera from Finland to analysis by Serokonversionskriterien.

The invention is illustrated by the following examples explained, but the invention is not limited to the examples is limited.

example 1 Identification of discriminatory Aritigen reactions by B. burgdorferi sensu lato a. Selection of the strain and the membrane fraction for determination of discriminatory antigens against anti-B. Burgdorferi antibody

A serological test system mostly becomes heterologous Responses determined because the one causing an infection Strain is unknown in most cases. Therefore, it is consequently, to choose a test strain that (i) if possible has many potential antigens and (ii) one if possible high reactivity in the reaction with antibodies from Human sera shows.

To determine potential antigens, membrane fraction II was obtained from two representatives of the species B. burgdorferi s. s (B31, HB1), B. afzelii, (Orth, Pko) and B. garinii (K15, W). For this purpose, grown cells were pelleted, the cells disrupted with a Vibrogen® cell mill, the lysate filtered and centrifuged again. The pellet, containing membrane fraction II, was washed with Tris / HCl, resuspended and the amount of protein determined. The membrane fraction II of the three species was analyzed using a modified SDS-PAGE according to Lämmli et al. (1970. Nature 227: 680-685) (stacking gel: 3% acrylamide / 2.6% bis-acrylamide, separating gel: 11% acrylamide / 1.8% bis-acrylamide, barrel length of the gel: 125 mm). The separated antigens were then transferred to an immobilizing membrane and stained with colloidal gold. A typical protein pattern of membrane fraction II of the strain Orth is shown in FIG. 1. The results of the subsequent counting of the potential antigens (stained with colloidal gold) are shown in FIG. 2. It shows that the representatives of the species B. afzelii and B. garinii have a higher number of potential antigens than the representatives of B. burgdorferi sensu stricto.

To determine the sensitivity, 86 sera from Lyme disease patients with ECM, NB, LA, ACA syndrome were tested against the above-mentioned representatives using surf blot (= modified Western blot) and the mean number of bands (one band corresponds to the formation of an antibody / antigen complex) for identified each type of syndrome. It was found that the representatives of B. afzelii and B. garinii had on average higher band numbers than the representatives of B. burgdorferi ss ( Fig. 2). When the representatives of B. afzelii and B. garinii were assessed in terms of the number of potential antigens and the average number of antibodies / antigen complexes (number of bands), the ORTH strain (B. afzelii) proved to be superior to the other strains tested in both criteria .

b. Testing representative patient sera using Vertical analysis (VA)

To determine the antigens significant for the Western blot vertical analysis, 670 human sera from three European regions were tested for their IgG and IgM reactivity against the strain ORTH (B. afzelii):

The vertical analysis was carried out according to a modified method by Towbin et al. (1979. Proc. Natl. Acad. Sci. USA 76: 4350-4354) using Western Surfblot. For this purpose, the proteins of a membrane fraction of the Orth strain according to Example 1 were separated using a 1-lane SDS gel and the antigens were blotted onto an immobilizing membrane. Then 1.5 wide strips were cut off from the right and left of the membrane and the strips were colored with Auro-Dye®. The middle part of the membrane was blocked with TBS-Tween 20-BSA and clamped in a surf blot apparatus. The human sera to be tested were filled into one track groove of the apparatus, incubated, washed with buffer and incubated with secondary antibody. For the development, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro-blue-tetrazolimchloride (NBT) were added as a substrate and the bands visualized. Fig. 3 shows a typical Western blot Surfblot pattern according to Testing with sera.

Fig. 4 shows the results of the vertical analysis of a selection of 37 human sera, tested against the membrane fraction II of the strain Orth. On the left are those 49 antigens (in decreasing size in kD) that were reactive with human serum antibodies in the various studies. In the listing, those antigens that were shown to produce cross-reactive or negatively discriminant reactions (= KRND reactions) were left out. The column next to the molecular weights indicates the names of those antigens which were detectable with known monoclonal antibodies ("E" means "equivalent to" and indicates with the following number the approximate molecular weight of that antigen that is detected by the monoclonal antibody in the homologous strain has been). The tested human sera are listed in the first line above; the line below the respective reaction table shows IgG reactions with "+", IgM reactions with "O". If an antigen was recognized by both an IgM and an IgG reaction of the antibodies of the same serum, these reactions were marked with a "⊗" symbol. Those antigens, the reaction of which with human serum antibodies are called discriminant, are shaded gray in the table. At the end of the table, the total number of IgG and IgM reactions of each serum is shown in two lines. The KRND reactions are not taken into account here either. The last line shows for each serum how it was classified according to the vertical analysis ("P" = positive, "-" = negative).

c. Statistical analysis to identify discriminatory Antigens

The aim of these evaluations was to generate antigenic reactions determine that a clear distinction between not enable seroconverted and seroconverted sera. Antigens that are of such analytical relevance must be discriminatory on the one hand (i.e. between the two Seroconversion states can distinguish), on the other hand they shouldn't be redundant (i.e. the response should not be associated with any other antigenic reaction).

In order to first differentiate between different and redundant antigen reactions, the surfblot results of each of the three sera panels from CZ / AT, Finland and Germany (670 sera in total) were evaluated using a redundancy analysis. The result of this analysis ( FIG. 5a) shows, depending on the sera panel, a total of 27 antigens that were identified as differentiating and not redundant.

In the analyzes carried out below, an attempt was made to with special attention to the results of the Redundancy analysis to capture the discriminating antigens.  

In the analyzes carried out below, an attempt was made to with special attention to the results of the Redundancy analysis to capture the discriminating antigens. Cross-reacting antigens were detected using sera from Syphilis patients against the strains Orth (B. afzelii), B31 (B. burgdorferi s. s.) and KL5 (B. garinii), each Membrane fraction II, were tested. In this sub-study representatives of all three species were selected because one broadest possible spectrum of cross-reacting antigens should be recorded. This investigation revealed for the Antigens 58.9 kDa (E60), 57.2 kDa (E58), 56.5 kDa and 47.7 kDa clear reactivity with antibodies to syphilis Patient. Therefore, these antigens, which are in the Redundancy analysis still shown as "differentiating" were excluded for further considerations.

In a parallel approach, discriminant antigen reactions were determined on the basis of the discriminant analysis: After the Czech / Austrian serum panel was most homogeneous in terms of its composition (the 130 human sera could be assigned to the three clinical phases of Lyme disease in approximately equal proportions), this was over analyzed the "Stepwise Logistic Regression". Of the total of 7 antigens that resulted from this study, the 58.9 kDa (E60) antigen had to be excluded from further studies because of its cross-reactivity (with syphilis sera, see above). Four (87.0 kDa, 41.2 kDa, 40.2 kDa, 25.1 kD), the remaining six antigens matched the other 27 antigens from the redundancy analysis. The two other antigens (28.0 kDa, 23.0 kDa) were only assessed as discriminatory by the discriminant analysis, but showed a relevant frequency (32.8-45.3% and 26.2-38.2%) when estimating the frequency of reactions (95% confidence interval estimate). According to the analysis criteria, an antigen only had a "relevant frequency" if the lower confidence limit was above a predetermined value of 20%. Two further antigens, 71.3 kDa and 70.4 kDa (both are contained in FIG. 5 of the 27 non-redundant antigens) were identified as "negatively discriminatory" by the discriminant analysis. The antigens 58.9 kDa (E60), 47.7 kDa, 71.3 kDa and 70.4 kDa are cross-reactive or negatively discriminatory and were therefore excluded.

Overall, by applying the Discriminant analysis the number of eligible Antigens from 27 to 29 (by the antigens 28.0 kDa and 23.0 kDa) increased. Through the cross-reaction study with syphilis sera and the negative discriminating antigens from the Discriminant analysis reduced this number to 6 now 23 antigens. Of these 23 antigens, 6 have already been confirmed by the discriminant analysis, with which 17 Antigens are examined for their analytical relevance had to. Since the antigen 52.4 kDa is not one for all sera specific reaction with IgG antibodies showed reduced the number of antigens to be examined continues to 16. These sixteen antigens were ranked on their frequency Reactions with antibodies from human sera were examined. The Estimation of frequency was made by calculating the 95% Confidence intervals reached. Also pointed out here Antigen response a "relevant frequency" when the Value of the lower confidence limit above a previous one fixed value of 20%.

In the two left-hand columns of FIG. 5, the 29 antigens which have been identified as differentiating and non-redundant are listed with their molecular weights and short names. The three subsequent columns contain information on each antigen in each of the three sera panels. Table fields marked with "+" indicate those antigenic reactions that have been identified as differentiating or non-redundant (via the redundancy analysis). With the exception of the two antigens 28.0 kDa and 23.0 kDa, at least one "+" label is entered for each antigen. However, the two antigens listed above were not identified using the redundancy analysis, but were determined using the discriminant analysis. The right column contains brief comments on the individual antigen reactions. The fields highlighted in gray indicate those reactions which, after all investigations, were of the highest analytical relevance for vertical analysis.

Those 10 antigens, the analytical relevance of which seemed most suitable for a vertical analysis, are listed with a gray background in FIG. 5.

Example 2: Horizontal analysis with OspC variants as reagent antigens a. Selection of Borrelia strains for horizontal analysis:

Sera from 56 seropositive Lyme disease patients were found tested against representatives of 35 OspC-RFLP types. This was done of 35 RFLP-type strains according to membrane fraction II Example 1 prepares the proteins using SD5-PAGE separated, transferred to immobilizing membranes and performed a horizontal analysis with human sera. 82% of Sera reacted with one or more of the 35 OspC Variants. Based on this study, the 20 am most frequently reacting variants are selected and are the Table 1 summarized.  

Table 1. OspC-RFLP types that react most often with Lyme disease sera

All OspC antigen RFLP types listed in Table 1 were subsequently used in horizontal analysis.

b. Detailed analysis of the anti-OspC immune response from Patients from different geographical regions:

Sera from 190 patients from the Åaland Islands, Finland, who suffer from Lyme disease, were tested by Western blot horizontal analysis ( Fig. 6) against the 20 most responsive variants of OspC protein (Table 1). The results are summarized in Fig. 7. The column labeled "Overall Reactions" shows the number of sera reacting with each strain. The RFLP type of the respective strain is also given. The OspC reaction pattern of each individual serum is divided into 3 types of reactions:

Total species reactions ("common" reactions): sera, the react with OspC variants of strains of all 3 species. Species sub-reactions ("shared" reactions): sera with OspC variants of strains from 2 of the 3 species react. Species-specific reaction (Species-specific): sera that react with OspC variants of only one species and therefore as Species-specific.

Strain-specific response (serotype-specific): the category the species-specific reactions were further subdivided in those sera that react with only one strain and are therefore considered to be strain-specific.

The category of specific reactions is the one that provides most of the information since it is the species indicates to which the infecting strain belongs. This Information can be used to predict the disease from be critical since there is a relationship between the disease syndrome the patient is suffering from and the Species of the infecting strain. B. afzelii can be found in are primarily isolated from skin and is associated with a chronic ACA skin syndrome. B. garinii strains isolated from CSF samples and are mostly associated with neuroborreliosis associated, while B. burgdorferi sensu stricto strains are primary associated with Lyme arthritis, particularly in North America is.

The number of sera falling into each of the reaction pattern categories is shown in Figure 7B . Approximately 78% of all sera show a reaction, the most prominent category being that of the "species-specific" (49% of the positive sera). It was also possible to report serotype specificity in 31% of the positive sera. The species-specificity of the sera of the "species partial reaction" and "species-specific reaction" categories is also shown in FIG. 7C. A predominance of reactions against OspC protein of the species B. afzelli and B. garinii was found in both categories. Within the "species-specific reaction" category there was an almost even distribution between the species. If one ascertains the distribution of these reactions among the individual OspC types within the corresponding species, in the case of B. afzelii almost all reactions for only 2 RFLP types, namely RFLP type 8 (strain H15) and RFLP type 22 ( Strain E51) was observed. In contrast, species-specific reactions of B. garinii OspC protein are evenly distributed and are found in 9 of 12 RFLP types. Fig. 6 shows a typical horizontal analysis with OspC variants tested against various human sera. The order of the Borrelia strains corresponds to that in FIG. 7.

c. Analysis of the anti-OspC humoral immune response by individual patients

The IgG and IgM response of individual patients was determined in the horizontal western blot analysis by testing the sera against the selected 20 OspC-RFLP types. Fig. 8 shows the reaction pattern of the IgG and IgM response of 37 patients, the part of a larger study of sera from 190 patients and 100 blood donors of AALAND Islands, Finland, was (Serenpanel according to Example 1). The study was carried out to test the sensitivity and specificity of horizontal western blot analysis in a highly endemic region. A positive IgG response was marked with "+" and a positive IgM response with "o". A positive IgG and IgM reaction is marked with "⊗".

In summary, the specificity of the IgG response of the individual patient is usually very specific, since it usually only reacts with one strain or is restricted to strains of a single species. In contrast, the IgM response is very broad and reacts with most of the OspC antigens tested. Fig. 8 shows the analysis of the Western blot analysis of the horizontal membrane fraction were used 20 strains representative of the three major species with appropriate patients' sera, which also according to the vertical analysis Example 1. II. The lower columns of Figure 8 indicate the "species specificity" of the IgG and IgM response for each individual serum.

d. Testing of recombinant OspC from different RFLP types (Horizontal analysis) in surfblot

In order to detect antibodies against individual membrane proteins in polyspecific sera, recombinant OspC protein from P. pastoris according to WO 94/25596 was used in this detection method, which was bound to nitrocellulose membrane. In contrast to conventional Western blot methods, the recombinant membrane protein is applied directly to the nitrocellulose membrane using a surf blot apparatus. With this method, 18 OspC types can be applied per blot. The test is also carried out with the surf blot apparatus (30 sera per blot). The blot is then incubated with a conjugate or secondary conjugated antibody and a substrate solution. The reaction pattern of a serum in line blot (horizontal analysis) is shown in FIG. 9.

e. Testing of recombinant OspC from different RFLP types (Horizontal analysis) in ELISA

Based on the ELISA, various recombinant OspC proteins tested against human sera as in Example 2d. The coupling of the recombinant OspC antigens to the Surface of the wells of a microtiter plate was made in a carbonate buffer (pH 9.6) at 4 ° C overnight. After this unspecific binding sites on the plastic surface were blocked (PBS, 10% FC5), the individual sera tested by aliquots of the sample with the bound antigen were incubated. To remove unbound protein or antibodies were washed several times and by one Test, e.g. B. alkaline phosphatase, conjugated antibodies proven. A control in which no antigen was added. A served as a positive control already tested positive serum or as a negative control a tested negative serum.

To carry out an ELISA assay according to the The present invention uses the following variants:

Variant A: Mixtures of all OspC-RFLP types are available a microtiter well is coupled.

Variant B: Mixtures of OspC-RFPL types derived from the same species, e.g. B. B. burgdorferi s. s., B. afzelii or B. garinii, are in one Microplate well coupled.

Variant C: Individual OspC RFLP types are divided into one Microplate well coupled.

Assay variant A is set up for a general screen positive reactivity used in horizontal analysis. Assay variant B can be used to switch between the general categories of reaction types in the To differentiate human immune response. The same applies here Criteria as described in Example 2b.

Assay variant C allows the extent to be assessed the cross-reactivity in the test sera and in such sera, which show an RFLP type strain specific immune response.  

This provides a direct statement about the species of the OspC RFLP-type of the infecting strain possible if necessary, also conclusions on the clinical picture of the Allows patients.

Example 3: Combination of vertical and horizontal analysis

The test results of the vertical and horizontal western blot analysis of 190 sera from patients from the Åaland island were evaluated together and are summarized in FIG. 10. In this representation, the individual forms of analysis (VA and HA) were compared with each other in terms of their efficiency. It is very clearly expressed that the combination of vertical and horizontal analysis has the highest detection rate.

The fields headed with the letters "VA" indicate the Values for the vertical analysis again, that with the letter "HA", that of horizontal analysis. The values by the Letter combination "V / H" are headed for the results of a combination of vertical and horizontal analysis. Positive sera are marked with "+" and negative sera marked with "-".

The fields highlighted in gray at the crossing points (fields that belong together are framed thick) of identical row and column headings indicate the absolute number and the percentage of sera, respectively. The fields headed with the letter "B" in FIGS . 10 and 11 represent the values of the band number criterion. "+" Stands for "positive" (≧ 4 bands IgG or ≧ 2 bands IgM), "/" for "borderline" (2-3 bands IgG or 1 band IgM) "-" for "negative" (0-1 Band IgG or 0 bands IgM), whereby the KRND antigen reactions were not included in the calculation.

FIG. 11 shows the results of the serum examples from FIGS . 4 and 8. Of the 37 sera in total, 33 sera were detected as seroconverted when the vertical and horizontal analysis were combined. Of these, nine sera could only be determined by horizontal analysis and two sera only by vertical analysis. Two sera showed no reaction when tested against membrane fraction II of the ORTH strain in the vertical analysis. A serum could nevertheless be determined to be positive via the horizontal analysis.

In Fig. 12 the results of the two examples for the horizontal (HA) and the vertical analysis (VA) are shown side by side. The human sera, their SYNTYPs (syndrome type) and the phase assignment can be found in the left column in the same order as in FIGS. 4 and 8. The three following column groups are titled "VA", "HA" and "V / H". The titles are short forms of vertical analysis (VA), horizontal analysis (HA) or a combination of both types of analysis (V / H). The column groups "VA" and "HA" have the same structure: The left column shows whether the serum in question was rated positive ("P") or negative ("-"). The three subsequent columns show how many reactions the respective serum had with IgG antibodies (+), IgM antibodies (O) or if both an IgG and an IgM reaction were directed against the same antigen (Ä). The column "V / H" shows the results of the combination of both types of analysis.

In the table, those sera that are either highlighted in gray only reported by the VA or only by the HA as positive were. In parallel, the band numbers are each other form of analysis.  

This table shows that of the 37 tested Sera only 22 positive from both VA and HA were evaluated. 11 sera were either only from the HA (9) or only recognized by the VA (2). In the combined Applying both evaluation criteria, 33 (89%) Sera detected. In the case of the two serums that are only available through the VA Both IgM and IgG could be determined Reactions. Of the nine, only through horizontal analysis determinable sera showed 4 sera exclusively one IgG, 2 sera only one IgM and 3 sera both an IgG as well an IgM reactivity.

The results of the three studies (CZR / Austria, Finland, Germany) are shown in comparison in FIG. 13. All sera (control and patient groups), a total of 670, of the three studies were assessed via vertical analysis. The sera from the control group, but also the patient sera from Finland and Germany, were also classified via the HA. In the patient sera of the CZR / Austria study, only those sera that were found to be "negative" in the VA were evaluated via the HA.

In the course of these analyzes, a very high prevalence in the Finnish control group (the Finnish sera come from the Åaland Islands - a highly endemic Region). To get the specificity values (from the results of the Control group does not calculate) the applied criteria falsify, 03187 00070 552 001000280000000200012000285910307600040 0002019629543 00004 03068 only the results of CZR / Austria and Germany studies as data basis accepted. When calculating the sensitivity, the Serenpanels from all three studies as a data basis be used. Only the sensitivity value of the Horizontal analysis in the CZR / Austria study had to should be left out, because in this study only those sera were used  the HA were rated negative via the vertical analysis had been diagnosed (see above).

If you carry out a combination of the two types of analysis, you will get values of 85% for both the specificity and the sensitivity, measured on the available sera panels. The detection rate of Lyme disease seroconverted sera, which were detectable with either the VA or HA, are shown in FIG. 14. Fig. 14 shows a direct comparison of the efficiency of FA and RA. It shows that on average about 72% of the sera rated as "positive" were recognized by both types of analysis. In addition, 29% of the sera were only detected by the HA and 9% of the sera only by the VA. In other words, only 91% of the sera would be detectable with the HA alone, and only 81% with the VA alone (always based on 100% through the combined application).

FIG. 15 shows a specificity of HA, namely that it is also possible to evaluate positively those sera which did not result in a single reaction with antigens of membrane fraction II of the ORTH strain. This table shows that about 37% of all these sera could still be found to be "positive" by HA. Based on the total number of all positive sera, this gives a share of 5%.

This is justified by the fact that one such a serological test performed only heterologous Reactions can be measured. In the early stages of a However, infection very often becomes antibodies against the OspC Antigen formed. However, since the antigen OspC is divided into several serological Groups are distinguishable and only one at the VA Variant is included (those that belong to the strain ORTH belongs), the sensitivity of the HA in these "non- Responder sera "higher.  

Example 4: Determination of seroconversion in early-stage patients

A vertical and horizontal analysis was performed with Lyme disease early-stage patients from the Finland study, since the proportion of early-phase sera was most common in this group. Fig. 16 shows a comparison of various forms of analysis in early stage patients. In the combined application of the VA and the HA, 50% more sera were recognized as positive than in the band number criterion. Overall, 75% of the early phase sera could be classified as "positive" when combined. Eleven of the 22 sera showed no reaction in the vertical analysis and were rated as "negative". Three of these 11 sera were found to be "positive" by the HA.

Claims (45)

1. Diagnostic agent, characterized in that it comprises as reagent components at least 4 of the antigens P870, P412, P402, P365, P315, P310, P280, P250, P230 and P180 derived from a membrane fraction of B. afzelii. 2. Diagnostic agent according to claim 1, characterized characterized it as a reagent component at least 6 of those from a membrane fraction of B. afzelii derived antigens P870, P412, P402, P365, P315, P310, P280, P250, P230 and P180. 3. Diagnostic agent according to claim 1, characterized characterized it as a reagent component at least 8 of those from a membrane fraction of B. afzelii derived antigens P870, P412, P402, P365, P315, P310, P280, P250, P230 and P180. 4. Diagnostic agent according to claim 1, characterized characterized that it is all of a Membrane fraction of B. afzelii-derived antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 includes. 5. Diagnostic agent according to claim 1 to 4, characterized characterized that the reagent components discriminating antigens that are specific to anti-B. burgdorferi antibodies react. 6. Diagnostic agent according to claim 1 to 5, characterized characterized that it is a membrane fraction of B. afzelii includes.   7. Diagnostic agent according to claim 1 to 5, characterized characterized in that the reagent components are recombinant Are antigens. 8. Diagnostic agent according to claim 1 to 4, characterized characterized that the reagent components were purified Are antigens. 9. Diagnostic agent according to one of claims 1 to 8, characterized in that the antigens on a solid supports are immobilized. 10. Diagnostic agent according to claim 9, characterized characterized in that the solid support is selected from Plates, strips, membranes, filters, paper, film or Pearls. 11. Diagnostic agent according to one of claims 1 to 10, characterized in that the antigens are derived are selected from a membrane fraction from B. afzelii from the group of B. afzelii Stamm Orth (DSM 11048) or strain H15 (DSM 11047). 12. Diagnostic agent according to one of claims 1 to 11 characterized in that it is hereinafter referred to as Reagent components contains at least one OspC antigen, which is essentially of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 are derived. 13. Diagnostic agent according to one of claims 1 to 11, characterized in that it is hereinafter referred to as Reagent components contains OspC antigens, which of all of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 are derived.   14. Use of a diagnostic agent according to one of the Claims 1 to 13 for the detection of Lyme disease. 15. Containing a test kit for the detection of Lyme disease

• a) a diagnostic agent according to any one of claims 1 to 13,
• b) substances for the detection of an antigen / antibody complex and
• c) an evaluation system for creating a serodiagnosis and instructions.

16. Test kit according to claim 15, characterized in that it is a surfblot apparatus or an analog working equipment includes. 17. Diagnostic agent, characterized in that it as reagent components at least 4 OspC antigens that Borreline RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 are derived. 18. Diagnostic agent according to claim 17, characterized characterized as being at least as reagent components 10 OspC antigens derived from Borreline RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 are included. 19. Diagnostic agent according to claim 17, characterized characterized as being at least as reagent components 15 OspC antigens derived from Borreline RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 are included. 20. Diagnostic agent according to claim 17, characterized characterized that it is used as reagent components OspC  Antigens from all of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 are included. 21. Diagnostic agent according to claim 17, characterized characterized that it comprises a membrane fraction, which of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 is derived. 22. Diagnostic agent according to claim 17 to 21, characterized characterized in that the reagent components are recombinant Are OspC antigens. 23. Diagnostic agent according to claim 17 to 21, characterized characterized that the reagent components were purified OspC antigens are from Borrelia. 24. Diagnostic agent according to one of claims 17 to 23, characterized in that the antigens to one solid carriers are bound. 25. Diagnostic agent according to claim 24, characterized characterized in that the solid support is selected from Sheets, strips, membranes, paper, film or pearls. 26. Use of a diagnostic agent according to one of the Claims 17 to 25 for the detection of Lyme disease and / or to determine the serotype of the Borrelia Pathogen. 27. Containing test kit for the detection of Lyme disease and / or determination of the serotype of the Borrelia pathogen

• a) a diagnostic agent according to any one of claims 17 to 26
• b) substances for performing detection of an antigen / antibody complex
• c) an evaluation system for creating a serodiagnosis and instructions.

28. Combination test kit containing a test kit according to Claim 15 and a test kit according to claim 27. 29. Immunoassay for the detection of an antibody against B. burgdorferi comprising the steps

• a) provision of a diagnostic agent which contains as reagent components at least 4 antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 derived from a membrane fraction of B. afzelii;
• b) Incubate a sample that may be anti-B. burgdorferi contains antibodies, with the diagnostic agent under conditions that allow the formation of an antibody / antigen complex, and
• c) determining the presence of anti-B. burgdorferi antibodies in the sample by detecting an antigen / antibody complex, in particular the complex formation of an antibody with one of the reagent components.

30. Immunoassay according to claim 29, characterized in that the reagent components discriminating antigens that are specific to anti-B. burgdorferi antibodies react. 31. Immunoassay according to claim 29 and 30, characterized characterized that the diagnostic agent a B. afzelii membrane fraction.   32. Immunoassay according to claim 29 and 30, characterized characterized in that the reagent components are recombinant Are antigens. 33. Immunoassay according to claim 29 and 30, characterized characterized that the reagent components were purified B, afzelii are antigens. 34. Immunoassay according to one of claims 29 and 30, characterized characterized that the biological sample is selected is made from animal or human liquids, such as Blood, plasma, serum, urine, cerebrospinal fluid and Synovial fluid. 35. Immunoassay for the detection of anti-B. burgdorferi antibodies and / or determination of the serotype of the Borrelia pathogen comprising the steps

• a) providing a diagnostic agent containing as reagent components at least 4 OspC antigens, derived from the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34,
• b) Incubate the sample, which may be anti-B. burgdorferi contains antibodies, with the diagnostic agent, and
• c) determining the presence of anti-B. burgdorferi antibodies in the sample by detecting an antigen / antibody complex, in particular the complex formation of an antibody with an OspC antigen component.

36. A method for serodiagnosis of Lyme disease comprising the steps

• a) providing a diagnostic agent containing as reagent components at least 4 antigens P870, P412, P402, P365, P315, P310, P280, P250, P230 and P180 derived from a membrane fraction of B. afzelii,
• b) Incubate a biological sample that may be anti-B. burgdorferi contains antibodies with the diagnostic agent and
• c) determining the presence of anti-B. burgdorferi antibodies in the sample by detection of an antigen / antibody complex, in particular the formation of a complex between an antibody and an antigen selected from the group of P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 is indicative of Lyme disease.

37. A method for serodiagnosis of Lyme disease comprising the steps

• a) providing a diagnostic agent containing as reagent components at least 4 OspC antigens, derived from the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34
• b) Incubate a biological sample that may be anti-B. burgdorferi contains antibodies with the diagnostic agent and
• c) determining the presence of anti-B. burgdorferi antibodies in the sample by detection of an antigen / antibody complex, in particular the formation of a complex between an antibody and an antigen selected from the group OspC antigens derived from the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 is indicative of Lyme disease.

38. The method according to claim 36 or 37, characterized characterized that the sample into a first or second Group is divided, the first group one Reaction between an antibody and at least one  Reagent component shows and thus in relation to Lyme Lyme disease is considered seroconverted, and the second Group has no reaction with any of the antigens. 39. The method according to claim 38, characterized in that in the first group a further determination of the Serotype of the Borrelia pathogen according to an immunoassay according to claim 35. 40. The method according to claim 38, characterized in that the sample of the second group according to an immunoassay one of claims 29 and 30 or according to claim 35 is subjected. 41. The method according to claim 40, characterized in that if necessary, the second group after a given one Time for another serodiagnosis according to a procedure according to any one of claims 36 to 40. 42. Monoclone antibodies against the immunologically reactive Antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 of a membrane fraction from B. afzelii. 43. Use of the monoclonal antibodies according to claim 42 in an immune enzyme test for the detection of Borrelia Antigens in a sample. 44. Use of the monoclonal antibodies according to claim 42 for the purification of immunologically reactive antigens from Borrelia. 45. Immunologically reactive antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 derived from B. afzelii strain Orth.